The 3′ end of most protein-coding genes and long non-coding RNAs is cleaved and polyadenylated. Recent discoveries have revealed that a large proportion of these genes contains more than one polyadenylation site. Therefore, alternative polyadenylation (APA) is a widespread phenomenon, generating mRNAs with alternative 3′ ends. APA contributes to the complexity of the transcriptome by generating isoforms that differ either in their coding sequence or in their 3′ untranslated regions (UTRs), thereby potentially regulating the function, stability, localization and translation efficiency of target RNAs. Here, we review our current understanding of the polyadenylation process and the latest progress in the identification of APA events, mechanisms that regulate poly(A) site selection, and biological processes and diseases resulting from APA.